There is considerable evidence that mammalian beta-tubulin is phosphorylated. Specifically, of the seven beta isotypes, the phosphorylated one is beta III, the isotype found almost entirely in neurons. The phosphate is added at a serine and perhaps a tyrosine near the C-terminus. All the evidence to date has been gathered by growth of cells and tissues in the presence of radioactive inorganic phosphate followed by tubulin isolation and determination of the labeled tubulin; thus, the actual extent of phosphorylation of beta III is unknown. Nor is it known if alpha-tubulin and the other beta isotypes are phosphorylated by a mechanism which would not be revealed by previous experiments. In addition, the role of tubulin phosphorylation is unknown. We have purified the alpha beta II-, alpha beta III-, and alpha beta IV-tubulin dimers from bovine brain and have determined their phosphate content chemically. We have found that alpha-tubulin is not phosphorylated and neither are the beta II or beta IV isotypes. However, beta III is phosphorylated with a stoichiometry of about 1.52 mol/mol. We have found that the phosphate on beta III is resistant to a wide variety of phosphatases except for human erythrocyte phosphatase 2A and that removal of the phosphate inhibits microtubule assembly in vitro stimulated by microtubule-associated protein 2 (MAP 2). However such an inhibition was not evident when microtubule assembly was induced in the absence of microtubule-associated proteins. Our results suggest the possibility that beta III phosphorylation may play a role in regulating microtubule assembly in vivo.